Some of the most wide-spread and powerful antithrombotic agents used in therapy are heparins and low molecular weigh active fragments of heparins. These agents have general drawbacks in their requirement of parenteral administration for therapeutic efficacy and in their rather unspecified anticoagulant activity. In the development of novel antithrombotic agents it is therefor highly desirable to find selective inhibitors of coagulation factors available with oral administration, e.g. low molecular weight compounds with a high biologic specificity. Initial efforts in field were directed to find inhibitors to thrombin, but since such inhibition might have clinical drawbacks, subsequent efforts have been directed towards the serine protease factor Xa, the enzyme directly responsible for thrombin activation. Factor Xa is an especially desirable target for the design of selective inhibitors in the development of anticoagulant drugs, since the two branches of the coagulation cascade (the intrinsic and extrinsic pathways) converge on this agent. A number of inhibitors of factor Xa are documented in the literature, such as the compound DX9065a and its analogues disclosed in EP 0 540 051 to Daiichi, which are selective to factor Xa compared to thrombin. For a review of synthetic factor Xa inhibitors, it is referred to Drugs of the Future, 1995, Vol. 20, No. 9, pp. 911-918: M Yamazaki. The structure of factor Xa is thoroughly discussed in J. Mol. Biol., 1993, Vol. 232, pages 947-66: K Padmanabhan et al, where it is confirmed that it comprises several epidermal growth factor (EGF)-like domains of which one is disordered in crystals. Generally, active factor Xa consists of a light chain comprising the EGF-domains and having a N-terminal region containing gamma-carboxyglutamic acid (Gla)-residues. The light chain is connected to a heavy chain by a disulfide bond and said heavy chain contains a catalytic domain featuring the active site triad of His236, Asp279 and Ser376 which has a high degree of similarity to other trypsine-like serine proteases. In this article, it is also reported certain difficulties to crystallize factor Xa inhibited with dansyl-Glu-Gly-Arg chloromethyl ketone (DEGR) possibly due to interactions of the C-terminal of the light chain and the active site. Also, in FEBS Letters, 1995, Vol. 375, pages 103-7: MT Stubbs et al., the difficulties in obtaining crystals of factor Xa for inhibition studies are acknowledged. The authors of this article have therefore instead attempted to investigate the binding of selected factor Xa inhibitors to the active site of trypsine to obtain structural information of the binding site for the inhibition of factor Xa. The coordinates from binding of DX9065a to trypsine are transferred to factor Xa, whereupon its hypothetical interaction with Factor Xa could be more closely studied. The recently published article in the Journ. of Biol. Chem. 1996, Vol. 271 (47), pp. 29988-92: H Brandsetter et al., discloses crystal structures of factor Xa deprived of the amino acids L1 -L44 (des-Gla-fXa) in complex with the previously mentioned inhibitor DX-9065a.
It would obviously constitute a major advantage to make factor Xa more available for inhibition studies in crystalline form in an with a retained active site, so a powerful model for designing inhibition candidates is obtained.